1. Field of the Invention
The present invention relates to a novel fluorescent label compound, which can be enzymatically incorporated into a nucleic acid. The labeled nucleic acid can be used to detect the presence of a DNA or RNA sequence of interest in a sample nucleic acid.
2. Description of the Related Art
The technique of hybridization of a labeled oligonucleotide to a sample DNA to afford sequence-specific nucleic acid detection has become a powerful tool in analytical molecular biology. Initially, labeling was accomplished mainly with radioactive isotopes. However, radioactive labeled probes typically have the disadvantage of instability, low resolution, and all the pitfalls of handling radioisotopes. In order to circumvent labeling with radioactive isotopes, a number of methods have been recently developed for non-radioactive labeling and detection.
For example, K. Muhlegger et al., "Synthesis and Use of New Digoxigenin-Labeled Nucleotides in Non-Radioactive Labeling and Detection-Labeled Nucleic Acids", Nucleosides & Nucleotides, 8 (5 & 6), pp. 1161-1163 (1989), describe the chemical synthesis and incorporation into DNA of novel digoxigenin-derivatized 5-aminoallyl-2'-deoxyuridine-5'-triphosphates. Hybridization and subsequent detection by ELISA technique allows the detection of homologous DNA down to 0.1 pg.
Hans-Joachim Holtke et al., "Non-Radioactive Labeling of RNA Transcripts In Vitro with the Hapten Digoxigenin (DIG); Hybridization and ELISA-Based Detection", Nucleic Acids Research, 18 (19), pp. 5843-5851 (1990), describe an alternative method for labeling nucleic acid probes with the cardenolide digoxigenin. However, the detection of digoxigenin disadvantageously requires a secondary reagent, such as an antibody linked to an enzyme.
EP-A2-63879 describes chemically stable new nucleotide derivatives that contain biotin, imino-biotin, lipoic acid and other determinants attached covalently to the pyrimidine or purine ring and their synthesis. These compounds will interact specifically and uniquely with proteins such as avidin or antibodies. This can be utilized for the detection and localization of nucleic acid components.
U.S. Pat. No. 4,617,261 discloses non-radioactive labeling reagents consisting of an alkylating intercalation moiety, such as a psoralen moiety, a divalent linker, and a monovalent label moiety, like biotin. These reagents are used to label nucleic acids.
Finally, Dirks et al., Experimental Cell Research, 194, pp. 310-315 (1991), describe the use of fluorescein-, digoxigenin- and biotin -(di)deoxyXTPs and terminal deoxynucleotidyl transferase for small scale labeling of synthetic oligonucleotides for use as probes in the in situ detection of multiple RNA sequences. Apparently, Boehringer Mannheim has now made available fluorescein-12 -dUTP for this purpose. See, BM Biochemica, 8 (5), p. 15 (September 1991).
Despite these known methods, there continues to exist a need for labels for nucleic acids which are easy to obtain, easy to incorporate into nucleic acid probes, and easy to detect.